The unloaded shortening velocity of skinned rabbit psoas muscle fibres is sensitive to [Ca²⁺]. To determine whether Ca²⁺ affects the unloaded shortening velocity via regulation of crossbridge kinetics or crossbridge number, the shortening velocity was measured following changes in either [Ca²⁺] or the number of active thin filament regulatory units. The native troponin C (TnC) was extracted and replaced with either cardiac TnC (cTnC) or a mixture of cTnC and an inactive mutant cardiac TnC (CBMII TnC). The unloaded shortening velocity of the cTnC-replaced fibres was determined at various values of [Ca²⁺] and compared with different cTnC:CBMII TnC ratios at a saturating [Ca²⁺]. If Ca²⁺ regulates the unloaded shortening velocity via kinetic modulation, differences in the velocity-tension relationship between the cTnC fibres and the cTnC:CBMII TnC fibres would be apparent. Alternatively, Ca²⁺ control of the number of active crossbridges would yield similar velocity-tension relationships when comparing the cTnC and cTnC:CBMII TnC fibres. The results show a decline in the unloaded shortening velocity that is determined by the relative tension, defined as the level of thin filament activation, rather than the [Ca²⁺]. Furthermore, at lower levels of relative tension, the reduction in unloaded shortening is not the result of changes in any cooperative effects of myosin on Ca²⁺ binding to the thin filament. Rather, it may be related to a decrease in crossbridge-induced activation of the thin filament at the level of the individual regulatory unit. In summary, the results suggest that Ca²⁺ regulates the unloaded shortening velocity in skinned fibres by reducing the number of crossbridges able to productively bind to the thin filament without affecting any inherent property of the myosin.